Construction of prokaryotic expression vector of chicken Prnp gene and expression in Escherichia coli

doi:10.3969/j.issn.1004-1524.2020.12.04

Acta Agriculturae Zhejiangensis ›› 2020, Vol. 32 ›› Issue (12): 2138-2146.DOI: 10.3969/j.issn.1004-1524.2020.12.04

• Animal Science • Previous Articles Next Articles

Construction of prokaryotic expression vector of chicken Prnp gene and expression in Escherichia coli

LI Xianchun¹(), LU Yan², MAO Yaofang¹, YANG Haifeng¹, YU Haishan¹, MA Yonghua1¹^,^*(), WAN Xuerui¹^,^*()

1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
2. State-Owned Assets Management Office, Northwest Normal University, Lanzhou 730070, China

Received:2020-04-09 Online:2020-12-25 Published:2020-12-25
Contact: MA Yonghua1,WAN Xuerui

Abstract

Abstract:

In order to construct the prokaryotic expression vector of chicken Prnp gene and express it in Escherichia coli, and to provide materials for the preparation of monoclonal antibodies against chicken prion protein, primers were designed according to chicken Prnp genome sequence reported by GenBank and the polyclonal site of pET-28a plasmid. Using healthy chicken whole blood genomic DNA as material, the Prnp gene of chicken was amplified by PCR. The target gene fragment was connected with pET-28a vector to construct recombinant prokaryotic expression vector. The recombinant bacteria were transformed into E. coli BL21(DE3) competent cells and induced by isopropyl-β-D-thiogalactoside (IPTG). The expressed recombinant protein was identified by SDS-PAGE. The results showed that the expression of recombinant protein was the highest at the final inducer concentration of 0.08 mmol·L ^-1, 16 ℃, 220 r·min^-1 for 7 hours. To sum up, the recombinant expression strain of pET-28a-ChPrnp was successfully constructed in this study, which provides a method for the study of the structure, physiological function and pathogenic mechanism of Prnp prion protein.

Key words: chicken, Prnp gene, recombinant expression vector, prokaryotic expression

CLC Number:

S831

LI Xianchun, LU Yan, MAO Yaofang, YANG Haifeng, YU Haishan, MA Yonghua1, WAN Xuerui. Construction of prokaryotic expression vector of chicken Prnp gene and expression in Escherichia coli[J]. Acta Agriculturae Zhejiangensis, 2020, 32(12): 2138-2146.

Figures/Tables 9

Table 1 Connection system

项目 Item	体积Volume/μL
项目 Item	1	2	3
pET-28a质粒片段pET-28a plasmid fragment	1.0	2.0	1.5
目的DNA Target DNA	7.0	6.0	6.5
10×连接酶缓冲液10×Ligase buffer	1.0	1.0	1.0
T4 DNA连接酶T4 DNA ligase	1.0	1.0	1.0
总体积Total volume	10	10	10

Table 2 Protein glue formula

试剂 Reagent	分离胶 Separating gel (12%)	浓缩胶 Concentrated gel(5%)
灭菌蒸馏水ddH₂O	3.3 mL	4.4 mL
30% ACR	4.0 mL	1.0 mL
1.5 mol·L^-1 Tris-HCl	2.5 mL	—
1.0 mol·L^-1 Tris-HCl	—	0.75 mL
10% SDS	0.1 mL	0.06 mL
10% APS	0.1 mL	0.06 mL
TEMED	10 μL	10 μL

Fig.1 PCR amplification product of target gene M, DL2000 DNA marker;1, PCR amplification product of ChPrnp(822).

Fig.2 Digested product of target DNA and pET-28a plasmid M, DL5000 DNA marker; 1, Digested product of target DNA; 2, Digested product of pET-28a plasmid.

Fig.3 PCR identification of recombinant plasmid M, DL2000 DNA marker; 1-3, PCR amplification products of bacteria solution 1, 2 and 3.

Fig.4 Double enzyme digestion analysis of recombinant expression vector M, DL5000 DNA marker; 1-3, Double enzyme digestion products of recombinant plasmids 1, 2 and 3.

Fig.5 Temperature optimization of induced expression of IPTG by recombinant bacteria 1-3, Protein products induced at 16, 28 and 37 ℃; M, Protein marker.

Fig.6 Optimization of concentration of IPTG induced by recombinant bacteria 1-5, Adding 0.08, 0.09, 0.10, 0.11, 0.12 mmol·L-1 IPTG, respectively; M, Protein marker.

Fig.7 Optimization of expression time of IPTG induced by recombinant bacteria M, Protein marker; 1-2, Expression products of recombinant bacteria induced for 5 and 7 h; 3, Uninduced recombinant bacteria.

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Construction of prokaryotic expression vector of chicken Prnp gene and expression in Escherichia coli

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